Studies on the active site of Pepsin c

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Abstract

Pepsin C, one of the minor gastric proteases of the pig, is fairlysimilar
in most of its properties to the major enzyme, pepsin. The most
marked difference between the two enzymes is the inability of pepsin C to
catalyse the hydrolysis of acetyl-phenylalanyl-diiodotyrosine, a good
synthetic substrate for pepsin. It was of interest to try to find which
group(s) in pepsin 0 was involved in the catalytic activity of the enzyme
and to make a comparison between this and the active site region of pepsin
itself.
The active site of pepsin G was investigated by the technique of
'affinity labelling1. Diazoacetyl norleucine methyl ester was found to
inactivate the enzyme very rapidly and irreversibly in the presence of
cupric ions at pH values above 4*5« The inactivation was specific, in
that one mole of inhibitor was incorporated per mole of enzyme when the
enzyme had lost 100$ of its activity. Evidence was found for the
existence of a binding site for the inhibitor and it was found that a
competitive inhibitor protected the enzyme against inactivation by the
diazo compound. The irreversible inhibitor did not interact to any great
extent with denatured pepsin C, all of which suggests that diazoacetyl
norleucine methyl ester inactivates the enzyme by reaction at the active
site.